Automated system for polynucleotide synthesis and purification

ABSTRACT

A method and system for polynucleotide synthesis and purification are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure squences caused by chain growth from extraneous support sites. Consequently, currently used rapid purification techniques depending on trityl hydrophobicity give a more highly purified product. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

FIELD OF THE INVENTION

The present invention relates generally to the synthesis andpurification of polynucleotides, and more particularly, to automatedtechniques for solid phase synthesis and purification of polynucleotidesusing phosphoramidite and/or hydrogen phosphonate chemistries.

BACKGROUND

A key factor in the recent advances in molecular biology has been thedevelopment of reliable and convenient methods for synthesizingpolynucleotides, e.g. Itakura, Science, Vol. 209, pgs. 1401-1405 (1980);and Wallace et al, pgs. 631-663, in Scouten, ed. Solid PhaseBiochemistry (John Wiley & Sons, New York, 1982). As the use ofsynthetic polynucleotides has increased, the demand for even greaterconvenience in the preparation of pure, ready-to-use polynucleotides hasalso increased. This demand has stimulated the development of manyimprovements in the basic procedures for solid phase synthesis, e.g.Sinha et al, Nucleic Acids Research, Vol. 12, pgs. 4539-4557(1984)(beta-cyanoethyl in phosphoramidite chemistries); Froehler et al,Tetrahedron Letters, Vol. 27, pgs. 469-472 (1986)(H-phosphonatechemistry); Germann et al, Anal. Biochem., Vol. 165, pgs. 399-405(1987); and Ikuta et al, Anal. Chem., Vol. 56, pgs. 2253-2256(1984)(rapid purification of synthetic oligonucleotides by way of tritylmoieties); Molko et al, European patent publication 241363 dated 3 April1987 (improved base-labile acyl protection groups for exocyclic amines),and the like.

In spite of such progress, difficulties are still encountered in currentmethods of polynucleotide synthesis and purification. For example,derivatized controlled pore glass (CPG), the current support of choicein most solid phase methodologies, can be responsible for spuriousindications of coupling yields, e.g. Pon et al, BioTechniques, Vol. 6,pgs. 768-775 (1988). Moreover, CPG, like most glasses, lacks chemicalstability in some of the highly corrosive deprotection reagents, such asconcentrated ammonia and trichloroacetic acid, used in polynucleotidesynthesis. As a consequence, the CPG support itself can be degraded inthe deprotection steps and can be a source of contamination of the finalproduct. This problem is exacerbated by the relative long reaction timesrequired to remove currently used protection groups for exocyclicamines. An extended period of deprotection is required to remove thesegroups after the polynucleotide has been cleaved from the solid phasesupport. Thus, complete automation of synthesis and purification hasbeen impractical. Another problem with CPG is that its surface supportschain growth at sites other than those associated with the 5' terminusof an attached nucleoside. Such "extraneous" chain growth gives rise toa heterogeneous population of 5'-blocked (usually tritylated)polynucleotides. Typically, the "extraneous" tritylated products lackone or more 3' nucleotides. This, of course, prevents one fromsuccessfully taking advantage of the relatively high hydrophobicity ofthe trityl group to purify "correct sequence" polynucleotides.Incorrect-sequence extraneous chains are also tritylated.

In view of the above, the field of solid phase polynucleotide synthesiscould be significantly advanced by the availability of alternativesupport materials which have the favorable mechanical properties of CPG,but which also possess greater chemical stability under the reactionconditions of polynucleotide synthesis, and which provide lessopportunity for extraneous chain growth during synthesis. The use ofsuch materials coupled with improved exocyclic protection groups wouldallow practical automation of polynucleotide synthesis and purificationin a single instrument.

SUMMARY OF THE INVENTION

The invention is directed to a system and method for automatedpolynucleotide synthesis and purification. Important features of theinvention include the use of a highly crosslinked porous polystyrenesupport for solid phase polynucleotide synthesis by H-phosphonate and/orphosphoramidite chemistries, and rapid on-line purification of thesynthesis product. The use of the polystyrene support significantlyreduces the number of failure sequences caused by extraneous initiationof polynucleotide chain growth on the support material. The polystyrenesupport also permits deprotection of the polynucleotide product in thepresence of the support material by virtue of its superior stability inthe presence of deprotection reagents.

Preferably, the exocyclic amines of the monomeric nucleosideintermediates are protected by acyl protection groups of the form--COCHR₁ R₂, wherein R₁ is H or lower alkyl, and R₂ is H, lower alkyl,lower alkoxy, lower aryloxy, or substituted-lower aryloxy. Preferably,the substituents of the lower aryloxy are electron-withdrawing, such asnitro-, cyano-, or sulfonate.

As used herein, the term lower alkyl refers to straight-chained,branched, or cyclic alkyls containing from 1 to 6 carbon atoms.Preferably, the term lower alkoxy refers to methoxy, ethoxy, isopropoxy,tertbutyloxy, or the like. Preferably, the term lower aryloxy refers tophenoxy, napthoxy, biphenyloxy, or the like.

"Electron-withdrawing" denotes the tendency of a substituent to attractvalence electrons of the molecule of which it is apart, i.e. it iselectronegative, March, Advanced Organic Chemistry, pgs. 16-18 (JohnWiley, New York, 1985).

These acyl protection groups, when used with the polystyrene supports ofthe invention, allow deprotection of the exocyclic amines and cleavageof the polynucleotide product from the solid phase support in a singlestep. Moreover, when beta-cyanoethyl protection groups are employed inthe phosphoramidite approach, deprotection of the internucleosidephosphates can also be effected. The single deprotection/cleavage stepoccurs rapidly enough so that the entire synthesis-purificationprocedure can be practically automated.

Preferably, the correct-sequence polynucleotides are purified from thecrude mixture cleaved from the reaction column by passage of the crudemixture over an adsorbent which preferentially adsorbs the 5'-blockinggroup of the correct-sequence polynucleotides. More preferably, wheneverthe 5'-blocking group is a trityl, the adsorbent is a nonswellablepolystyrene solid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrammatically represents a preferred apparatus forimplementing the method of the invention.

FIG. 2 illustrates an autoradiogram of two 18-mer oligonucleotidesyntheses, one on CPG (lanes 1 and 2, crude and purified, respectively)and the other on polystyrene (lanes 3 and 4, crude and purified,respectively).

DETAILED DESCRIPTION OF THE INVENTION

The invention is a method and system for producing purifiedoligonucleotides and/or polynucleotides of a predetermined sequence. Themethod comprises the steps of (i) providing a nonswellable porouspolystyrene support, (ii) providing a 5'-blocked protected nucleosidehaving a 3'-hydroxyl and a 5'-hydroxyl, the 5'-blocked protectednucleoside being attached to the nonswellable porous polystyrenesupport, usually by a base-labile linkage between the 3'-hydroxyl of theprotected nucleoside and the support, such that the 5'-blocked protectednucleoside forms a protected correct-sequence chain; (iii) deblockingthe 5'-hydroxyl of the correct-sequence chain; (iv) reacting with the5'-hydroxyl of the correct-sequence chain a 5'-blocked protectednucleoside monomer selected from the group consisting of 5'-blockedprotected nucleoside-3'-phosphoramidites and 5'-blocked protectednucleoside-3'-H-phosphonates to form either a protected correct-sequencechain or a failure sequence, the failure sequence having a 5'-hydroxyl;(v) capping the failure sequence by reacting a capping agent with the5'-hydroxyl of the failure sequence; (vi) repeating steps (iii)-(v)until the polynucleotide of the predetermined sequence is obtained;(vii) deprotecting the exocyclic amines of the polynucleotide andcleaving the polynucleotide from the nonswellable porous polystyrenesupport to form a cleavage mixture; and (viii) purifying thepolynucleotide from the cleavage mixture. Preferably, the step ofpurifying includes the steps of exposing the cleavage mixture to ahighly crosslinked polystyrene adsorbent, separating the blockedpolynucleotide from the failure sequences by washing the polystyreneabsorbent to preferentially remove the failure sequences, deblocking the5'-hydroxyl of the polynucleotide, and eluting the polynucleotide fromthe polystyrene adsorbent. Preferably, the step of reacting includes thestep of oxidizing the internucleoside phosphorous from the trivalent tothe pentavalent state in the phosphoramidite chemistry. Preferably, themethod includes the further step of oxidizing the internucleotidephosphorous from the trivalent to the pentavalent state prior to thestep of deprotecting in the H-phosphonate chemistry.

As used herein, the term polynucleotide refers to a single strandedchain of either deoxyribonucleotides or ribonucleotides having from afew, e.g. 2-20, to many, e.g. 20 to several hundred or more,nucleotides. The term also includes chains of nucleosides linked byanalogs of the phosphate bond, e.g. thiophosphates, and the like.

Detailed procedures for the phosphoramidite and hydrogen phosphonatemethods of polynucleotide synthesis are described in the followingreferences, which are incorporated by reference: Caruthers et al, U.S.Pat. Nos. 4,458,066 and 4,500,707; Koester et al, U.S. Pat. No.4,725,677; Matteucci et al, J. Amer. Chem. Soc., Vol. 103, pgs.3185-3191 (1981); Caruthers et al, Genetic Engineering, Vol. 4, pgs.1-17 (198 ); Jones, chapter 2, and Atkinson et al, chapter 3, in Gait,ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press,Washington, D.C., 1984); Froehler et al, Tetrahedron Letters, Vol. 27,Pgs. 469-472 (1986); Garegg et al, Tetrahedron Letters, Vol. 27, pgs.4051-4054 and 4055-4058 (1986); Froehler et al, Nucleic Acids Research,Vol. 14, pgs. 5399-5407 (1986); Usman et al, J. Am. Chem. Soc., Vol.109, pgs. 7845-7854 (1987 ); Froehler, Tetrahedron Letters, Vol. 27,pgs. 5575-5578 (1986) and Andrus et al, Tetrahedron Letters, Vol. 29,pgs 861-864 (1988).

An important feature of the invention is the use of a nonswellableporous polystyrene support for synthesis. As used herein, "nonswellable"means that the porous polystyrene material remains substantiallymechanically rigid, in particular does not increase in volume, whenexposed to solvents, reactants, and products of the phosphoramiditeand/or hydrogen phosphonate chemistries. Mechanical rigidity isdesirable for efficient transfer of reagents to the polynucleotide chainduring synthesis. Nonswellability of the porous polystyrene dependsdirectly on the degree of crosslinking among the styrene polymers. Suchcrosslinking is usually measured by a crosslink ratio, which is themolar ratio of crosslinking material (e.g. divinylbenzene) and chainmaterial (e.g. styrene). Preferably, the nonswellable porouspolystyrenes of the invention have a crosslink ratio in the range ofabout 40-60 percent, and more preferably, they have a crosslink ratio ofabout 50 percent.

As used herein, "porous" means that the nonswellable polystyrenecontains pores having substantially uniform diameters in the range ofbetween 100-4000×10⁻⁸ cm. Preferably, the pore diameters are about1000×10⁻⁸ cm. Several different means are available for manufacturingporous polystyrene solids. As used herein, the term "porous" includes,but is not limited to, so-called macroporous polystyrenes and so-calledmacroreticular polystyrenes. These materials are widely availablecommercially in a variety of shapes and sizes, e.g. PolymerLaboratories, Ltd. (Shropshire, United Kingdom); Hamilton Co. (Reno,NV), or the like. Preferably, the nonswellable porous polystyrenesupports are used in the form of beads having a diameter in the range of15-100×10⁻⁴ cm, and more preferably, in the range of 50-70×10⁻⁴ cm.

Prior to its use in synthesis, the nonswellable porous polystyrene mustbe linked to a 5'-blocked protected nucleoside, which forms the firstnucleoside of the polynucleotide to be synthesized. The nature of thislinkage, the 5'-blocking agent, and the protecting groups of theexocyclic amines and internucleoside phosphorous are important featuresof the invention. Preferably, the first 5'-blocked protected nucleosideis linked to the polystyrene support by way of a base-labile linkage.More preferably, this linkage is an aminomethylsuccinate group, as iscommonly used in phosphite triester synthesis, e.g. Atkinson et al, pgs.45-49, in Gait, ed. Oligonucleotide Synthesis: A Practical Approach (IRLPress, Oxford, 1984). The linkage is formed by reacting a 5'-blockedprotected nucleoside-'3-O-succinate with an amino-derivatizedpolystyrene support. Synthesis of the 5'-blocked protectednucleoside-3'-O-succinate is well known in the art e.g. Atkinson et al,pgs. 47-49 (cited above); and U.S. Pat. No. 4,458,066. Accordingly,these references are incorporated by reference.

As used herein, "5'-blocked" refers to a group attached to the5'-hydroxyl of either the monomeric nucleoside intermediates used in theinvention, or the correct-sequence chain of protected nucleosides.(However, note that chains initiated at "extraneous" sites can be5'-blocked and yet not be of the correct sequence). Selection of the5'-blocking group is constrained by three criteria: (i) it must mask the5'-hydroxyl of the monomer so that it does not compete with the5'-hydroxyls of the correct-sequence chains during the monomer additionsteps, (ii) it must be acid-labile, in that it can be removed so as toexpose the 5'-hydroxyl upon mild acid treatment, and (iii) it must besufficiently hydrophobic to allow 5'-blocked correct-sequence chains tobe preferentially adsorbed onto a polystyrene adsorbent over unblockedfailure sequences. Preferably, the 5'-hydroxyls are protected as tritylethers. That is, the 5'-hydroxyl blocking agent, or group, is a trityl.Most preferably, the blocking group is 4,4'-dimethoxytrityl. Synthesisof 5'-blocked nucleoside intermediates is well know in the art, e.g.Froehler et al (cited above), and Caruthers et al (U.S. patents citedabove). The trityl blocking groups are removed, that is, the5'-hydroxyls are "deblocked" by exposure to a mild protic acid. Severaltrityl deblocking reagents have been used in solid phase polynucleotidesynthesis, e.g. Caruthers et al, Genetic Engineering, Vol. 4, pgs. 1-17(1984). Preferably, deblocking is accomplished by exposure to 2%trichloroacetic acid for about 3 minutes at room temperature.

As used herein, "protected" in reference to the monomeric nucleosideintermediates and the correct-sequence chains means (i) that theexocyclic amines of either compound have been acylated with a protectiongroup that prevents the exocyclic amine from participating in thesynthetic reaction steps, and (ii) that the internucleoside phosphorousof phosphoramidite intermediates are masked by a base-labile protectiongroup. Preferably, the phosphorous protection group is beta-cyanoethyl,as disclosed by Koester et al, U.S. Pat. No. 4,725,677. Many acylprotection groups are available for use in the invention, e.g. Jones,pgs. 23-34, in Gait, ed. (cited above). Preferably, the exocyclic amineprotection groups are sufficiently base-labile so that thecorrect-sequence chains can be deprotected and cleaved from thepolystyrene support in the same reaction step. As mentioned above, thepreferred protection groups are those disclosed by Molko et al (citedabove). Briefly, the groups are attached to the monomeric nucleosideintermediates by acylation of the exocyclic amino groups of the bases(adenine, guanine, and cytidine) of the deoxynucleosides2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytosine, or of theribonucleosides, adenosine, guanosine, and cytosine. Thymidine anduridine do not need base protection. Acylation occurs by reacting theacid chloride, such as methoxyacetyl chloride, isobutyryl chloride, orphenoxyacetyl chloride, or the acid anhydride, such as methoxyaceticanhdride, isobutyric anhydride, or phenoxyacetic anhydride, with the3',5' protected nucleosides. The 3' and 5' protecting groups can beeither trimethylsilyl or dimethoxytrityl. The trimethylsilyl groups areapplied with either hexamethyldisilazane or trimethylsilyl chloride andcan be removed under mild conditions with a neutral aqueous treatment.The dimethoxytrityl group can be applied with dimethoxytrityl chlorideeither before or after acylation. After acylation protection of theamino group and 5' dimethoxytritylation, the 3' hydroxyl group isconverted to a phosphoramidite moiety. This phosphitylation is achievedtypically with bis(diisopropylamino)methoxyphosphine orbis(diisopropylamino)cyanoethoxyphosphine with catalysis bydiisopropylammonium tetrazolide to give the methyl or cyanoethylphosphoramidite nucleosides, respectively, as shown by Formula I.##STR1##

Here, R₁ and R₂ are as described above. DMT represents dimethoxytrityl.B represents adenine, guanine, thymidine, or cytidine. R' representsmethyl or beta-cyanoethyl. And iPr is isopropyl. With the aboveprotection group, deprotection and cleavage can be achieved by treatmentin concentrated (29%) ammonia for 6 hours at 20° C., or for 1 hour at55° C.

As used herein, "correct-sequence chain" refers to a chain ofnucleosides which is capable of reacting with an additional monomericnucleoside intermediate via its 5'-hydroxyl (i.e. it is uncapped) andwhose sequence corresponds to that of the desired polynucleotide. Theterm includes the first nucleoside attached to the solid phase support(i.e. a nucleoside chain of one unit) as well as the completedpolynucleotide product of the predetermined sequence. As used herein,"failure sequence" refers to chains of nucleosides which have notreacted with a monomeric nucleoside intermediate during an addition stepand which are subsequently capped. The term also includes polynucleotidechains whose growth was initiated at an extraneous site of the solidphase support.

Thiophosphate analogs of polynucleotides can be synthesized inaccordance with the invention following the thionization steps taught byFroehler, Tetrahedron Letters, Vol. 27, 5575-5578 (1986), forH-phosphonate chemistry, or the thionizaton steps taught by Stec et al,J. Am. Chem. Soc., Vol. 106, pgs. 6077-6079 (1984), for phosphoramiditechemistry.

The polystyrene support is amino-derivatized by standard procedures,e.g. Wallace et al, pgs. 638-639, in Scouten, ed. Solid PhaseBiochemistry (cited above). Briefly, hydroxymethylpthalimide is reactedwith the polystyrene support with a catalytic amount of methylsulfonicacid to form pthalimidomethyl polystyrene. This material is treated withhydrazine to remove the pthalimide protecting group and giveaminomethylated polystyrene. The amino loading varies from 20-60 umolesof amino functionality per gram of nonswellable porous polystyrene. Thislevel can be controlled by adjusting the concentrations of the reagentsand reaction time.

This material is then reacted with the 5'-blocked protectednucleoside-3'-O-succinate. Unreacted amine of the polystyrene arerendered inactive by acylation with a moncarboxylic acid, e.g. asdisclosed in U.S. Pat. No. 4,458,066. Preferably, the amines arerendered inactive by acetylation with acetic anhydride.

As outlined above, synthesis of the desired polynucleotide usuallyproceeds by repeated cycles of deblocking, monomer addition, and cappinguntil synthesis is complete. As used herein, the term capping refers toreacting either the free 5' hydroxyl of a 3' to 5' growing nucleotidechain or the free 3' hydroxyl of a 5' to 3' growing nucleotide chainwith a capping agent to render the chain incapable of participating insubsequent condensation steps. The preferred capping agents of theinvention are phosphite monoesters of the form: ##STR2## wherein R,either alone or together with the oxygen to which it is attached, isunreactive with the reagents used in solid phase oligonucleotidesynthesis, particularly phosphoramidites or nucleoside hydrogenphosphonates. Preferably, R represents a lower alkyl, anelectron-withdrawing substituted lower alkyl, a lower alkyl- orhalo-substituted aryl, or a heterocycle containing nitrogen, oxygen, orsulfur and from 5-8 carbon atoms. More preferably, R is methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl,cyclopentylmethyl, isopentyl, neopentyl, n-hexyl, neohexyl, isohexyl,cyclohexylmethyl,beta-cyclopentylethyl, lower alkyl- or halo-substitutedphenyl, lower alkyl- or halo-substituted benzyl, or lower alkyl- orhalo-substituted phenylethyl, morpholinyl, thiomorpholinyl, piperidinyl,piperazinyl, beta-electron-withdrawing-substituted ethyl, or the like.In further preference, the electron-withdrawing substituent ofbeta-electron-withdrawing-substituted ethyl is cyano, nitro,phenylsulphonyl, or phenylester. Most preferably, thebeta-electron-withdrawing-substituted ethyl is beta- cyanoethyl. Infurther preference, the lower alkyl- or halo-substituents of the loweralkyl- or halo-substituted phenyl and benzyl are methyl, chloro, orbromo. In further preference, morpholinyl, thiomorpholinyl, andpiperidinyl are morpholino, thiomorpholino, and piperidino,respectively.

The chemical structures illustrated by Formula II are referred to in theliterature as both phosphites and phosphonates. Reflecting theapproximate usage in the literature, the structures will be referred toherein as phosphites, except when R is a nucleoside. In such cases thestructure will be referred to as a hydrogen or H- phosphonate.

As illustrated by Formula III, the capping method of the inventioncomprises reacting a phosphite monoester defined by Formula II, 1, withthe free 5' or 3' hydroxyl of a failure sequence, 2, in the presence ofa sterically hindered acid chloride, 3, to form a phosphite diester, 4,between the failure sequence and a group which is inert to subsequentreaction steps. ##STR3##

Preferably, the capping agents of the invention (1 in Formula IV below)are prepared by alkaline hydrolysis of the symmetrical phosphitediesters, 5, as described by Gibbs et al in Synthesis, pgs. 410-413(1984), which is incorporated by reference. The phosphite monoester 1can be used directly as a salt after evaporating volatile by products ofthe reaction or after purification by conventional means. ##STR4##

In the sterically hindered acid chloride 3, R' is preferably tert-butyl,sec-butyl, cyclohexyl, adamantyl, norbornyl, phenyl, aryl, or the like.More preferably, R' is tert-butyl, norbornyl, or adamantyl. Mostpreferably, R' is adamantyl.

Preferably, X⁺ is ammonium, lower alkylammonium, pyridinium, lutidinium,cyclohexylammonium, 1,8-diazabicyclo[5.4.0]undec-7-ene ammonium, a metalsalt cation such as Na⁺, K⁺, Li⁺, Ba⁺, Mg⁺, or the like. Morepreferably, X⁺ is triethylammonium, tetrabutylammonium,diisopropylethylammonium, pyridinium, lutidinium, or cyclohexylammonium.Most preferably, X⁺ is triethylammonium, tetrabutylammonium, or1,8-diazabicyclo[5.4.0]undec-7-ene ammonium.

Preferably, prior to delivery to the synthesis column bearing theoligonucleotide, a phosphite monoester of the invention and its cationiccounter ion are dissolved in a solution comprising an aprotic polarsolvent, such as acetonitrile, tetrahydrofuran, dichloromethane, or thelike, or some combination thereof, and a mild base such as pyridine,picoline, lutidine, collidine, or the like. Pyridine is the mostpreferred mild base. Preferably, the concentration of the phosphitemonoester is between about 0.01 to 0.10 molar. Likewise, the stericallyhindered acid chloride (3 in Formula III), prior to delivery to thesynthesis column, is dissolved in a solution comprising an aprotic polarsolvent, such as acetonitrile, tetrahydrofuran, dichloromethane, or thelike, or some combination thereof, and a mild base such as pyridine,picoline, lutidine, collidine, or the like. Pyridine is the mostpreferred mild base. The respective solutions are delivered concurrentlyto the synthesis column bearing the growing oligonucleotide so that themolar ratio of the phosphite monoester to the sterically hindered acidchloride present in the reaction mixture is about 1:5. This operationcan be readily performed by an automated DNA synthesizer, such as theApplied Biosystems models 380A, 380B, or 381A. The capping procedure ofthe invention is performed as a step in each cycle, after the couplingreaction, to render the failure sequences inert. Preferably, thesynthesis column is immersed in the reaction mixture for about 20-120seconds at room temperature, after which the reagents are flushed fromthe column with a solvent, such as acetonitrile, tetrahydrofuran,dichloromethane, pyridine, or the like, or some combination thereof. Allvessels within the instrument must be maintained rigorously free ofmoisture and oxygen under an atmosphere of an inert gas, such as argon.

At the completion of synthesis, the polynucleotide chains (bothcorrect-sequence chains and failure sequences) are deprotected andcleaved from the polystyrene support to form a cleavage mixture.Preferably, this is accomplished by treatment with concentrated ammoniumhydroxide for 4-5 hours at room temperature or for about 1 hour at 55°C. The cleavage mixture is applied to a solid phase adsorbent thatpreferentially adsorbs the 5'-blocked deprotected polynucleotides. Thepreferential adsorption is accomplished by selecting a blocking groupand solid phase adsorbent so that they have a strong mutual attactionrelative to the rest of the polynucleotide. The attraction can be basedon hydrophobicity, charge, or other physiochemical property. Tritylblocking groups are preferentially adsorbed onto polystyrene on thebasis of hydrophobicity. Preferably, whenever trityl blocking groups areused the solid phase adsorbent is a nonswellable porous polystyrenepretreated with a concentrated alkylammonium salt solution. Preferably,the nonswellable porous polystyrene used as the adsorbent is used in theform of beads having a diameter in the range of about 15-100×10⁻⁴ cm andpore size in the range of 100-1000 angstroms. More preferably, the beaddiameter is in the range of 50-70×10⁻⁴ cm and pore diameter is about 300angstroms.

Prior to applying the cleavage mixture to the polystyrene adsorbent, theadsorbent is flushed with an organic solvent, such as acetonitrile, towet the surface, then a concentrated alkylammonium salt solution,preferably a 2 M solution of triethylamine acetate, is applied toestablish a lipophilic counterion. After the cleavage solution,containing the polynucleotide synthesis mixture in concentrated ammonia,is applied to the polystyrene adsorbent. The adsorbent is then washed,preferably with dilute ammonia (a 3% solution) and water, to remove thefailure sequences and other contaminants, the correct-sequence chainsare deblocked, i.e. de-tritylated, and the de-tritylated polynucleotideis eluted from the adsorbent. Preferably, the elution is accomplishedwith a neutral solvent, e.g. 20% acetonitrile in water, or like solvent.

Preferably, the method of the invention is automated. The apparatus forautomating can take several forms. Generally, the apparatus comprises aseries of reagent reservoirs, a synthesis chamber containing thenonswellable porous polystyrene support, a purification chamber (whichmay be the same or different from the synthesis chamber) containing thesolid phase adsorbent, and a computer controlled means for transferringin a predetermined manner reagents from the reagent reservoirs to andfrom the synthesis chamber and the purification chamber, and from thesynthesis chamber to the purification chamber. The computer controlledmeans for transferring reagents can be implemented by a general purposelaboratory robot, such as that disclosed by Wilson et al, BioTechniques,Vol. 6, pg. 779 (1988), or by a dedicated system of tubing, andelectronically controlled valves. Preferably, the computer controlledmeans is implemented by a dedicated system of valves and tubingconnecting the various reservoirs and chambers. In further preference,the reagents are driven through the tubing by maintaining a positivepressure in the reagent reservoirs by means of a pressurized inert gas,such as argon, as is used by many widely available automatedsynthesizers, e.g. Applied Biosystems, Inc. models 380B or 381A DNAsynthesizers.

A diagrammatic representation of a preferred embodiment of such anapparatus is illustrated in FIG. 1. The apparatus of FIG. 1 is set forthas if the phosphoramidite chemistry were being employed. The sameinstrument can also be used to automate the Hphosphonate synthesis andpurification with obvious modifications, e.g. different synthesisreagents are used, different reaction times are required, and the like.These modifications are readily implemented via programmable controller48, or like means. 5'-blocked protected nucleoside intermediates arestored in reservoirs 2 through 10, one reservoir each for the fournatural nucleosides. Optionally, an additional reservoir 10 is providedfor a 5'-blocked protected nucleoside analog, e.g. deoxyinosine, alinking agent, e.g. U.S. Pat. No. 4,757,141, or like intermediates. Thereservoirs 2 through 10 containing the synthesis intermediates areconnected to synthesis chamber 28 by way of valve block 24 whoseoperation is controlled by controller 48. Synthesis reagents are storedin reservoirs 12 through 18. For example, in phosphoramidite chemistrythese can be 12 trichloroacetic acid in dichloromethane for deblocking,13 iodine/lutidine/water/tetrahydrofuran solution for oxidizinginternucleoside phosphorous, 14 tetrazole/acetonitrile solution foractivating the nucleoside intermediates, 15 ammonium hydroxide forcleaving the completed chain from the synthesis support, 161-methylimidazole/tetrahydrofuran solution and 17tetrahydrofuran/lutidine/acetic anhydride solution for capping, and 18acetronitrile for washing. These reagent reservoirs are connected tosynthesis chamber 28 by way of valve block 22 which is controlled bycontroller 48. Synthesis proceeds under programmed control with eachnucleotide being added to the growing chain by successive cyclesdeblocking, addition, capping, and oxidizing. Reagents removed fromsynthesis chamber 28 are directed to either trityl collection station30, waste reservoir 38, or purification chamber 34 by way of valve block32, which is controlled by controller 48. During each cycle tritylblocking groups released by deblocking are monitored photometrically attrityl collection station 30 to track the efficiency of the synthesis.

When synthesis is complete, the synthesis support is treated withconcentrated ammonium hydroxide to deprotect and cleave thepolynucleotide chains. Before the resulting solution (the cleavagesolution) is transferred to purification chamber 34, the polystyreneadsorbent in the chamber is flushed first with acetonitrile and thenwith triethylammonium acetate, from reservoirs 40 and 41, respectively.The cleavage mixture is transferred to purification chamber 34 via valveblock 32 where it reacts with the polystyrene adsorbent. The polystyreneadsorbent is then treated with a series of purification reagents fromreservoirs 40 through 46 to separate failure sequences and impuritiesfrom correct-sequence polynucleotides and to elute the correct-sequencepolynucleotides from the adsorbent. Transfer of reagents from thereservoirs and to and from the purification chamber 34 are made by wayof valve blocks 26 and 36, which are controlled by controller 48 First,dilute ammonium hydroxide from 40 flushes purification chamber 34 toremove capped failure sequences and other contaminants. Next, thetritylated polynucleotides are detritylated by treatment with a 2%triflluoroacetic acid solution in water, reservoir 44, and the thedetritylated polynucleotides are eluted with a 20% acetonitrile solutionin water, reservoir 42, and are collected in product vessel 50.

The following examples serve to illustrate the present invention. Theconcentrations of reagents, temperatures, and the values of othervariable parameters are only to exemplify the invention and are not tobe considered limitations thereof.

EXAMPLES Example I Comparison of Extraneous Chain Initiation on CPG andPolystyrene Supports During Synthesis

A 5'-tritylated-thymidine-derivatized polystyrene support (4000 angstrompore size), a 5'-tritylated-thymidine-derivatized CPG support (1000angstrom poresize), and a 5'-tritylated-thymidine-derivatized CPGsupport (500 angstrom pore size) were treated with5'-trityldeoxyadenosine-3'-phosphoramidite (A) and tetrazole activatorfor one hour and then detritylated. The nucleosidic material was thenremoved by treating the supports with ammonia, and separately analyzedby HPLC, the pertinent results of which are shown in Table I. Additionof A to the supports can only occur at two types of sites: (i) reactivesites on the support surface which were never capped or became uncapped,or (ii) detritylated thymidine. Reaction at the former sites leads tothe detection of adenosine monophosphate (AMP) in the HPLC analysis, andin practice to a heterogeneous population of tritylated polynucleotidescleaved from the synthesis support (as discussed below). Reaction at thelatter sites leads to the detection of an A-T dinucleotide in the HPLCanalysis. The presence of the latter sites reflects instability of thetrityl group during storage. Other compounds detected in the HPLCanalysis are thymidine (the predominant component) and benzamide, aby-product of the removal of the protecting group of adenine.

                  TABLE I                                                         ______________________________________                                        Relative Amounts From Chromatogram Peaks                                      Support  AMP     thymidine  benzamide                                                                             A-T dimer                                 ______________________________________                                        Polystyrene                                                                            1.9%    --         --      0.4%                                      CPG      5.7%    --         --      0.8%                                      (1000                                                                         angstrom)                                                                     CPG      4.1%    --         --      2.4%                                      (4000                                                                         angstrom)                                                                     ______________________________________                                    

The data indicate that the polystyrene support generates less than halfthe extraneous start sites as does CPG. Therefore, polynucleotides madewith the polystyrene support are more pure and can be more easilypurified than polynucleotides made on a CPG support.

Example II Synthesis and Purification of an 18-mer Oligonucleotide onCPG and Polystyrene: Comparison of Tritylated Failure Sequences

Two 18-mer oligonucleotides of the same sequence were synthesized on anApplied Biosystems model 380B synthesizer programmed for usingphosphoramidite chemistry, one on a CPG support and the other on apolystyrene support in accordance with the invention. Eacholigonucleotide was radiolabelled by ³² P-phosphorylation. The productof each synthesis was analysed by polyacrylamide gel electrophoresisbefore and after purification. FIG. 2 is an autoradiogram of the gelafter the components of the purified and unpurified products wereseparated. Lanes 1 and 2 correspond to the unpurified and purifiedoligonucleotide synthesized on CPG, respectively. Lanes 3 and 4correspond to the unpurified and purified oligonucleotide synthesized onpolystyrene in accordance with the invention. In both cases, theproducts were purified using a polystyrene adsorbent in accordance withthe invention. In particular, polystyrene adsorbent (50 milligrams per 5OD units of oligonucleotide to be purified) was packed in a purificationchamber, e.g. a standard flow through chromatography column, with aninternal volume of about 500 ul. The polystyrene adsorbent waspre-washed with 5 ml of acetonitrile, then flushed with 5 ml of 2 Mtriethylammonium acetate. The cleavage mixture was slowly forced throughthe purification chamber manually with a 5 ml syringe at about 2 ml perminute to load the tritylated sequences. After loading, the purificationchamber was washed first with 10 ml of 3% ammonia/water and then with 10ml water. 3 ml of 2% trifluoroacetic acid in water was applied to thepurification chamber and allowed to set for 3 minutes, afterwhich 15 mlof water was flushed through the chamber to remove residual acid.Finally, the purified oligonucleotide was eluted from the adsorbent with1 ml of 20% acetonitrile/water.

Table II gives the relative amounts of the correct-sequencepolynucleotides and the major failure sequences in the four productsbased on a densitometric analysis of the autoradiogram. It is clear thateven though the CPG synthesis results in a purer crude product, thepolystyrene synthesis provides a significantly greater yield afterpurification because the population of tritylated polynucleotides in itscrude product is far more homogeneous than that of the CPG synthesis.

                  TABLE II                                                        ______________________________________                                        Densitometer Data                                                                     CPG      CPG       Polystyrene                                                                            Polystyrene                                       Crude    Purified  Crude    Purified                                  Sequence                                                                              Lane 1   Lane 2    Lane 3   Lane 3                                    ______________________________________                                        18-mer   59.6%   69.5%      36.0%    84.4%                                    17-mer  9.2      11.0      4.2      3.2                                       16-mer  4.5      3.9       3.1      1.3                                       15-mer  3.7      1.0       3.1      0.5                                       14-mer  2.3      0.1       4.3      0.1                                       13-mer  2.3      1.0       4.9      0.6                                       12-mer  1.8      0.9       7.3      0.1                                       ______________________________________                                    

The foregoing disclosure of preferred embodiments of the invention hasbeen presented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdisclosed. Obviously many modifications and variations are possible inlight of the above teaching. The embodiments were chosen and describedin order to best explain the principles of the invention and itspractical application, to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

We claim:
 1. A method for synthesizing a polynucleotide of apredetermined sequence, the method comprising the steps of:(a) providinga nonswellable porous polystyrene support, remaining substantiallymechanically rigid during synthesis; (b) providing a 5'-tritylatedprotected nucleoside, the 5'-tritylated protected nucleoside beingattached to the nonswellable porous polystyrene support, such that the5'-tritylated protected nucleoside forms a protected correct-sequencechain; (c) detritylating the correct-sequence chain to free a5'-hydroxyl; (d) reacting with the 5'-hydroxyl of the correct-sequencechain a 5'-tritylated protected nucleoside monomer selected from thegroup consisting of 5'-tritylated protectednucleoside-3'-phosphoramidite and 5'-tritylated protectednucleoside-3'-hydrogen phosphonate to form either a protectedcorrect-sequence chain or a failure sequence, the failure sequencehaving a 5'-hydroxyl; (e) repeating steps (c) and (d) until thepolynucleotide of the predetermined sequence is obtained; (f) cleavingthe polynucleotide from the nonswellable porous polystyrene support toform a cleavage mixture, the cleavage mixture containing tritylatedpolynucleotide and untritylated failure sequences; and (g) purifying thepolynucleotide by exposing the cleavage mixture to a hydrophobicadsorbent, washing the hydrophobic adsorbent to preferentially removeuntritylated failure sequences, detritylating the 5'-hydroxyl of thetritylated polynucleotide, and eluting the untritylated polynucleotidefrom the hydrophobic adsorbent.
 2. The method of claim 1 wherein saidstep of cleaving includes deprotecting said polynucleotide.
 3. Themethod of claim 2 further including the step of capping said failuresequence by reacting a capping agent with said 5'-hydroxyl of saidfailure sequence.
 4. The method of claim 3 wherein said hydrophobicadsorbent is a nonswellable polystyrene adsorbent.
 5. The method ofclaim 3 wherein said 5'-tritylated protected nucleoside monomer is5'-tritylated protected nucleoside-3'-hydrogen phosphonate, and whereinsaid step of capping occurs after said step of repeating.
 6. The methodof claim 3 wherein said 5'-tritylated protected nucleoside monomer is5'-tritylated protected nucleoside-3'-phosphoramidite, and wherein saidstep of capping occurs after said step of reacting.
 7. The method ofclaim 6 wherein said 5'-tritylated protectednucleoside-3'-phosphoramidite is defined by the formula: ##STR5##wherein: R₁ is selected from the group consisting of hydrogen and loweralkyl;R₂ is selected from the group consisting of hydrogen, lower alkyl,lower alkoxy, lower aryloxy, cyano-, nitro-, or sulfono-substitutedlower aryloxy; R' is selected from the group consisting of methyl andbeta-cyanoethyl; DMT is dimethoxytrityl; and B is selected from thegroup consisting of adenine, guanine, thymidine, and cytidine.
 8. Themethod of claim 7 wherein R₁ is selected from the group consisting ofhydrogen, methyl, ethyl, and propyl, and wherein R₂ is selected from thegroup consisting of hydrogen, methyl, ethyl, propyl, methoxy, ethoxy,isopropoxy, tert-butyloxy, phenoxy, napthoxy, and biphenoxy.
 9. Themethod of claim 8 wherein R₁ is selected from the group consisting ofhydrogen and methyl, and wherein R₂ is selected from the groupconsisting of hydrogen, methyl, ethoxy, and phenoxy.
 10. The method ofclaim 4 wherein said nonswellable polystyrene adsorbent is treated withan alkylammonium salt solution prior to said step of purifying.